Evaluation of multicomponent mixtures using modulated light beams

An apparatus for analyzing sample material having more than one fluorescing species, said apparatus comprising:

• a first light source emitting a first excitation light beam;
• a second light source emitting a second excitation light beam;
• means for modulating the intensity of said first excitation light beam according to a first periodic function of time;
• means for modulating the intensity of said second excitation light beam according to a second periodic function of time different from said first periodic function of time;
• a sample container in which sample material is presented;
• optical means after said first and second beam modulators for directing said first and said second excitation light beams to said sample material in said sample container for interaction with said sample material;
• a photosensitive detector receiving fluorescence light emitted by said sample material in response to interaction with said first and second excitation light beams, said detector providing signal information representative of the intensity of received light;
• an optical element in front of said detector for excluding excitation light from reaching said detector;
• and
• means connected to said detector for processing said signal information to extract a first component signal corresponding to said first excitation light beam and a second component signal corresponding to said second excitation light beam.

A flow cytometer comprising:

• a first light source emitting a first excitation light beam;
• a second light source emitting a second excitation light beam;
• means for modulating the intensity of said first excitation light beam as a first periodic function of time;
• means for modulating the intensity of said second excitation light beam as a second periodic function of time different from said first periodic function of time;
• a flow cell through which said sample material flows;
• optical means after said first and second beam modulators for directing said first and said second excitation light beams to said sample material in said sample container for interaction with said sample material;
• and
• a photosensitive detector receiving fluorescence light emitted by said sample material in response to interaction with said first and second excitation light beams, said detector providing aggregate signal information representative of the intensity of received light, wherein said aggregate signal information includes a first component signal corresponding to said first excitation light beam and a second component signal corresponding to said second excitation light beam.

A method of analyzing sample material having more than one fluorescing species, said method comprising the steps of:

• providing a plurality of intensity-modulated excitation light beams, each of said plurality of excitation light beams being modulated at a respective unique frequency;
• directing said plurality of intensity-modulated excitation light beams to interact with said sample material;
• detecting fluorescence emission light from said sample material to provide signal information representative of detected light intensity versus time;
• and
• extracting a plurality of component emission signals from said signal information, wherein each of said plurality of component emission signals corresponds to a respective one of said plurality of excitation light beams.